Fig. 2.

Loss of function analysis. (A) Splice-blocking MO design. The sequences of MO-1 and MO-2 and their target sites at the 3′-end of pak4 exon 4 are shown. Two MOs were necessary for complete knockdown due to the unmasking of a cryptic splice donor. F1, R1 and R2 refer to PCR primers used to monitor splicing (see Section 2). The red arrows indicate the positions of in-frame stop codons in the abortively spliced mRNA. (B) Translation-blocking MO of the indicated sequence targeting a site upstream from the start codon (black circle). (C) RT-PCR analysis of splice-blocking. Embryonic RNA from the indicated stages was isolated after injection of the control MO (ctl) or combined splice-blocking MOs (S) and used as template for PCR. The 300-bp PCR product was amplified by the primer pair F1 + R2 and represents the correctly spliced pak4 mRNA, while the 600-bp product (primers F1 + R1) represents the abortively spliced mRNA. The 600-bp product appeared at tailbud stage (TB), indicating pak4 transcription from the zygotic genome. At this stage the 300-bp product had declined in splice-inhibited embryos and had disappeared by the 5-somite stage (5s). This pattern persisted through the final stage tested, 5 dpf. As a positive control a fragment of β-actin was amplified in the corresponding samples. (D) MO effectiveness. Pak4 protein was detected by immunoprecipitation/western blotting from equal numbers of embryos injected with the splice MOs (S; MO-1, MO-2) or a mixture of splice- and translation-blocking MOs (M; MO-3), or from uninjected embryos (U). Equal protein input for immunoprecipitations was confirmed by BCA assays. At shield stage the splice MOs reduced pak4 partially, while the splice + translation-blocking mixture had a much stronger effect. Both treatments gave essentially complete inhibition at 24 hpf, although some residual pak4 protein was detected in the splice morphants. Ponceau S staining of the IgG heavy chain band (IgG) is shown below as a control for immunoprecipitation recovery. (E) Representative embryos at various stages injected with control (Ctl), splice-blocking MOs (Splice; 1.5 + 1.5 ng), or a combination of the translation- (3 ng) and splice-blocking (1.5 + 1.5 ng) MOs (Splice + MO-3). Only the latter showed disrupted morphology, as observed at 24 hpf and steadily worsening through 5 dpf. Scale bar, 250 μM. (F) Summary of pak4 mRNA rescue data at 24 hpf. Morphant embryos were classified as normal, moderate (bent axis, small head and eyes), or severe (kinked and shortened axis, small head and eyes, unidentified forebrain and hindbrain, or missing head and tail – “monster”). The proportion and severity of affected embryos were both decreased substantially by co-injection of 900 pg pak4 mRNA.